Genes for the biosynthesis of the fungal polyketides hypothemycin from Hypomyces subiculosus and radicicol from Pochonia chlamydosporia
Genes for the biosynthesis of the fungal polyketides hypothemycin from Hypomyces subiculosus and radicicol from Pochonia chlamydosporia
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DOI：
10.1128/aem.00478-08
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Kealey, James T.
中科院分区：
文献类型：
--
作者：
Reeves, Christopher D.;Hu, Zhihao;Kealey, James T.
对亚cullosus和衣孢Pochonia chlamydosporia真菌多酮素hypothemycin和radicicol的生物合成基因簇进行了测序。这两个簇编码一个还原聚酮合成酶（PKS）和一个非还原PKS，如玉米赤霉烯酮簇中的PKS，以及具有PKS后功能的酶。将o -甲基转移酶（OMT）敲除构建体引入到H. subiculosus中，导致菌株的4- o -去甲基hypothemycin产量增加，但由于H. subiculosus的转化非常困难，我们选择使用异源基因表达来表征hypothemycin的生物合成。在体外，OMT可以甲基化缺乏4- o -甲基的各种底物，黄素依赖的单加氧酶（FMO）可以环氧化具有1‘,2’双键的底物。谷胱甘肽s-转移酶催化了hypothemycin的7‘,8’双键的顺-反异构化。在酵母中表达两个hypothemycin PKS基因（但不单独表达）可产生反式7′，8′-脱氢玉米赤霉烯醇（DHZ）。在菌株中表达OMT，表达FMO，表达细胞色素P450，分别导致DHZ甲基化，1',2'-环氧化和羟基化。自由基基因簇编码卤化酶和细胞色素P450同源物，它们被认为分别催化氯化和环氧化。提出了次霉素和根尖醇的生物合成方案。上述两个簇编码的pks与玉米赤霉烯酮簇编码的pks合成的产物不同，但它们具有显著的序列同一性。这些PKS可能为探索真菌PKS编程机制提供了一个有用的系统。
Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia, respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae, plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4-O-desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1',2' double bond. The glutathione S-transferase catalyzed cis-trans isomerization of the 7',8' double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans-7',8'-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1',2'-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.